Interaction of Synthetic Human SLURP-1 with the Nicotinic Acetylcholine Receptors

Human SLURP-1 is a secreted protein of the Ly6/uPAR/three-finger neurotoxin family that co-localizes with nicotinic acetylcholine receptors (nAChRs) and modulates their functions. Conflicting biological activities of SLURP-1 at various nAChR subtypes have been based on heterologously produced SLURP-1 containing N- and/or C-terminal extensions. Here, we report the chemical synthesis of the 81 amino acid residue human SLURP-1 protein, characterization of its 3D structure by NMR, and its biological activity at nAChR subtypes. Radioligand assays indicated that synthetic SLURP-1 did not compete with [125I]-α-bungarotoxin (α-Bgt) binding to human neuronal α7 and Torpedo californica muscle-type nAChRs, nor to mollusk acetylcholine binding proteins (AChBP). Inhibition of human α7-mediated currents only occurred in the presence of the allosteric modulator PNU120596. In contrast, we observed robust SLURP-1 mediated inhibition of human α3β4, α4β4, α3β2 nAChRs, as well as human and rat α9α10 nAChRs. SLURP-1 inhibition of α9α10 nAChRs was accentuated at higher ACh concentrations, indicating an allosteric binding mechanism. Our results are discussed in the context of recent studies on heterologously produced SLURP-1 and indicate that N-terminal extensions of SLURP-1 may affect its activity and selectivity on its targets. In this respect, synthetic SLURP-1 appears to be a better probe for structure-function studies.

Various recombinant versions of SLURP-1 have been shown to modulate nAChRs, mostly of the α7 subtype 7,8,12 , but with contradictory results due to the expressed SLURP-1 proteins containing additional C-or N-terminal fusion tags. For example, Chimienti and colleagues reported potentiation of α7 nAChR-mediated currents by a recombinant myc-His 6 -SLURP-1 fusion construct at low nanomolar concentrations 12 . In contrast, recombinant SLURP-1 expressed in E. coli, containing an additional methionine residue at the N-terminus (hereafter referred to as rSLURP-1), exhibited inhibitory activity at α7 nAChR (at micromolar concentrations) 13 .
To resolve these conflicting data on SLURP-1 activities, we report here the chemical synthesis and biological activity of the 81 amino acid human SLURP-1 identical in amino acid sequence to the human serum-derived protein. Using a combination of solid phase peptide synthesis and native chemical ligation 14 , high purity protein was obtained in multi-milligram amounts sufficient for structural and functional studies. The synthetic protein was characterized by HPLC, MS, and NMR which confirmed the three-finger fold structure. Most importantly, our pharmacological data revealed for the first time the interaction of synthetic SLURP-1 (sSLURP-1) with several neuronal nAChR subtypes.

Results
Human SLURP-1 synthesis and NMR structural analysis. Human SLURP-1, with 81 amino acid residues and five disulfide bonds, is considerably larger than most three-finger proteins from snake venoms (up to 62 residues and four disulfides). It is reminiscent of the classical long-chain α-neurotoxins, which are typically composed of up to 75 residues with a 5 th disulfide bond in the central loop II. In contrast, in all Ly6 proteins, including SLURP-1, the 5 th disulfide resides in the N-terminal loop I 4 . Given the size of the target SLURP-1 molecule, we resorted to a peptide segment ligation approach to overcome the size limitation of traditional stepwise solid phase peptide synthesis (SPPS) (Figs 1 and 2) 15 . Accordingly, the SLURP-1 polypeptide chain was split into three segments, which were individually assembled by either Boc or Fmoc SPPS (see Materials and Methods). Thiazolidine-4-carboxylic acid (Thz) was used in place of Cys21 16,17 to prevent cyclisation and oligomerisation during the first chemical ligation of SLURP-1  and SLURP-1[51-81]. Following cleavage from the solid support and purification, the segments were joined via native chemical ligation in one-pot fashion as described previously 16,18 .
The fully reduced SLURP-1 polypeptide was obtained in good yield (69% based on the limiting starting peptide segment SLURP-1[51-81]). Folding and disulfide formation of the synthetic molecule was achieved using protocols described recently for inclusion body refolding of rSLURP-1 produced in E. coli 13,19 . The folding kinetics and the overall HPLC folding profile were essentially identical to those reported for rSLURP-1 and allowed preparation of synthetic SLURP-1 in high purity and in multi-milligram quantities (Fig. 2D-F). High resolution MS analysis indicated a monoisotopic mass of 8837.1 ± 0.1 Da, in excellent agreement with the theoretical monoisotopic mass of 8837.02 Da demonstrating formation of five disulfide bonds (Fig. 2F).
To verify the anticipated three-finger fold of the synthetic material, we performed NMR experiments under the same conditions as reported previously for rSLURP-1 (i.e., H 2 O/D 2 O (9:1), pH 4.8, 310 K) 19 . The natural abundance 1 H-15 N HSQC spectrum of SLURP-1 showed good dispersion of amide proton and nitrogen resonances (Fig. 3A) suggesting that the molecule adopts a well-defined three-dimensional structure. Overall, the spectrum is highly similar to that of rSLURP-1 19 . Two-dimensional TOCSY and NOESY spectra were used to assign backbone amide and CαH protons. Comparison of the Hα chemical shift of each residue obtained from synthetic SLURP-1 spectra to the values available for rSLURP-1 (BMRB ID: 25225 and 25226; Fig. 3B) revealed excellent agreement, suggesting the proteins have highly similar three-dimensional structures. This suggestion is further supported by several key long-range NOEs observed in the NOESY spectra of synthetic SLURP-1, including Lys2Hα-Arg20Hα, Cys28Hα-Cys51Hα, Met29Hα-Cys71Hα, Thr30Hα-Arg49Hα and Leu76Hα-Tyr4Hδ/ε, all consistent with the proposed three-finger fold. Taken together, these data confirm that our synthetic SLURP-1 has a tertiary structure similar to that of rSLURP-1.
Human SLURP-1 does not compete with α-Bgt at human neuronal α7, muscle-type nAChRs and AChBPs. The recombinant form of SLURP-1 was shown previously to displace bound α-Bgt from the muscle-type nAChR of Torpedo californica and the mollusk Lymnaea stagnalis AChBP 13 . However, in the present study, the synthetic version of SLURP-1 did not compete with α-Bgt for either proteins (Fig. 4). In addition, no competition with α-Bgt binding was observed at either human (h) α7 nAChR or Aplysia californica AChBP (Fig. 4).

Discussion
Human SLURP-1 has the canonical three-finger folded structure of the snake α-neurotoxins, which are known as potent antagonists of nAChRs 4 . SLURP-1 has been shown to participate in a number of cellular regulation pathways, supposedly by acting on the homomeric α7 nAChR subtype [6][7][8][9]21,22 . However, the mechanism of interaction between SLURP-1 and α7 nAChR remains unclear due to the disparities in the activities of the recombinant human SLURP-1 constructs used. Recently, it was demonstrated that rSLURP-1 inhibited α7 nAChR-mediated currents 13 , whereas potentiation was reported previously for the myc-tagged fusion protein 12 . In contrast, binding of rSLURP-1 at the orthosteric and allosteric sites of different targets was registered 13 , which is in agreement with the similar activity of ws-Lynx1, thus supporting the proposed binding models 23,24 . We suspected these discrepancies potentially originate from the different chemical structures of the various recombinant forms of SLURP-1 used. Hence to resolve the conflicting data, we chemically synthesized the SLURP-1 protein (sSLURP-1) identical in amino acid sequence to the naturally-occurring human molecule 5 , and determined its structural and biological properties.
Three-finger proteins have been obtained previously by stepwise chemical solid phase peptide synthesis (SPPS) 25 . However, to the best of our knowledge, this approach has been limited to selected short-chain α-neurotoxins typically comprising ~60 amino acids and four disulfide bonds, with the longest being the synthetic non-conventional neurotoxin, built of 66 residues with five disulfides 26 . Larger proteins, including SLURP-1, are generally difficult to synthesize by stepwise Fmoc or Boc SPPS alone 15 .
Chemical synthesis of SLURP-1 was achieved using a convergent approach whereby the polypeptide was divided initially into three shorter peptide segments, each ranging in size of about 20-30 amino acids. The segments were prepared by Boc or Fmoc SPPS in good yield and purity, purified individually by HPLC and chemo-selectively linked together using the recently established one-pot native chemical ligation protocol 16 . Folding and disulfide bond formation of the synthetic 81-mer was achieved over 72 h using a glutathione redox shuffling system. To our knowledge, the successful chemical synthesis of SLURP-1 reported here, is the first example of a long chain three-finger protein of the Ly6 family obtained solely through chemical synthesis.
To unambiguously confirm the anticipated 3D structure, we performed NMR experiments that allowed near-complete assignment of backbone NH and CαH protons (as well as partial side chain proton assignments). This analysis and comparison with data previously obtained for rSLURP-1 19 , established that the synthetic molecule is structurally highly comparable to the recombinant protein.
Contrary to earlier reports of various recombinant SLURP-1 versions interacting with AChBPs and nAChRs 6-9,19,21 , we did not observe competition of synthetic SLURP-1 with radio-iodinated α-Bgt for binding to AChBPs of L. stagnalis and A. californica, nor to muscle-type T. californica nAChR (Fig. 4). Interestingly, although neither synthetic SLURP-1 and rSLURP-1 did compete with α-Bgt binding to hα7 nAChR, only rSLURP-1 inhibited ACh-evoked currents at hα7 nAChR with an IC 50 of ~1 μM and the inhibition was enhanced with increasing ACh concentrations 13 . However, synthetic SLURP-1 did not inhibit either hα7 or rα7 nAChRs in the presence of low and high concentrations of ACh ( Fig. 5A-C). The antagonistic effect of sSLURP-1 was only observed under the influence of the α7 nAChR positive allosteric modulator PNU120596, where substantial inhibition of hα7-mediated epibatidine-induced Ca 2+ influx (Fig. 5D) was observed.
Screening of sSLURP-1 at 10 μM against a number of heteromeric human neuronal nAChR subtypes, demonstrated preferential inhibition of nAChRs co-expressing α3 and β4 subunits ( Fig. 6A and B), with hα3β4 nAChR being more sensitive to inhibition by sSLURP-1 compared to hα3β2 and α4β4 nAChRs. This finding suggests that the binding site of sSLURP-1 might be located at the interface of α3 and β4 subunits and sSLURP-1 behaved as a competitive antagonist of the hα3β4 subtype. On the other hand, both hα9α10 ( Fig. 6A and B) and rα9α10 ( Fig. 6A and C) nAChRs showed ACh-dependent sSLURP-1 inhibition, with sSLURP-1 exerting its action at relatively high ACh concentrations. Ws-Lynx1 (a recombinant version of Lynx1 lacking the GPI anchor) was also shown to profoundly inhibit hα7, α3β2, and α4β2 nAChRs 23 , and the chimeric α7/glycine (Gly) receptor 24 in similar fashion. Overall, sSLURP-1 inhibition at the human αβ (except hα4β2) and α9α10 nAChRs subtypes can be registered at low ([ACh] ~EC 50 ) and high ACh concentrations, respectively, conditions that are more "physiologically relevant". In contrast, the action of sSLURP-1 on the hα7 nAChR is observed only in the presence of the artificial potentiator PNU120596.
Taken together, the results obtained for α7 and α9α10 nAChRs suggest that sSLURP-1 behaves as a 'silent' negative allosteric modulator, exerting its inhibitory effect at the nAChRs only when the receptor channels are in a stable open state. A similar unmasking effect on ligand activity was also reported for α-conotoxin MrIC 27 and the marine sponge-derived 6-bromohypaphorine 28 , where both behave as an agonist by eliciting concentration-dependent increases in [Ca 2+ ] i via PNU120596-modified hα7 nAChR.
As for α9α10, the mode of action of sSLURP-1 on this nAChR subtype mirrors the proposed negative allosteric mechanism of rSLURP-1 13 and ws-Lynx1 23,24 where both are more potent inhibitors of α7 nAChR in the presence of high ACh concentrations. Despite the structural similarities in the NMR-Hα chemical shift profiles of sSLURP-1 and rSLURP-1 (Fig. 3B), these proteins clearly behave differently at their targets.
Using the published NMR structure of rSLURP-1 (PDB ID: 2MUO), we built a model for sSLURP-1 (Fig. 7A) to investigate in more detail the structural differences between the two proteins. Molecular dynamic simulation of both structures indicates that the additional N-terminal methionine (Met0) residue in the rSLURP-1 is tightly packed inside the disulfide-rich core of the molecule (known to be important for stabilizing the overall conformation), whereas the absence of this residue in sSLURP-1 may allow Arg20 to protrude from the protein surface, possibly allowing it to participate in receptor interactions ( Fig. 7B and C). Furthermore, Lys2 and Asp75 form a stable salt-bridge, which is absent in rSLURP-1 (Fig. 7C, blue arrow). Such structural changes may account for the different biological actions observed for sSLURP-1 and rSLURP-1.
Recently, another member of the Ly6 protein superfamily, SLURP-2, was recombinantly expressed in E. coli with an additional N-terminal methionine residue, similar to rSLURP-1 29 . In previous publications utilizing different fusion forms, SLURP-2 was claimed to act selectively on α3-containing nAChRs 30 . However, rSLURP-2 is functionally more similar to ws-Lynx1 than to rSLURP-1. At micromolar concentrations, rSLURP-2 inhibited α4β2, α3β2 and α7 nAChRs, whereas at lower concentrations it potentiated the α7 nAChR-mediated currents 29 . This study also provided the NMR structure for rSLURP-2 (PDB ID: 2N99) revealing a considerable conformational mobility, comparable to that earlier observed for rSLURP-1. Although we did not perform a direct comparison of the various reported recombinant forms of SLURP-1, our results suggest that even one additional methionine residue at the N-terminus, probably by affecting the spatial structure, can produce marked changes in the functional activity of Ly6 proteins. showing that the positively charged Arg20 is now more solvent exposed. Arrows show regions occupied by Met0 residue in rSLURP-1 or solvent-accessible area in sSLURP-1. (C) Superimposed rSLURP-1 (light brown) and sSLURP-1 (cyan) structures showing the differences in the "head" region where Met0 is located in rSLURP-1. The N-terminal amino groups are indicated by red arrows. In the sSLURP-1 structure, a salt-bridge between residues Lys2 and Asp75 is present (blue arrow). Some residues have been omitted for clarity.

Conclusions
We wish to emphasize that our work on the synthetic protein identical in amino acid sequence to the naturally-occurring human SLURP-1 does not undermine the previous work on Ly6 analogs produced in E. coli. The activities reported with them may open new avenues to diagnostics and drug development. However, our results clearly show that unraveling physiologically-relevant mechanisms for endogenous regulators requires the study of compounds which should be as close as possible to the native proteins. In this context, total chemical synthesis has been particularly instrumental in the past for small proteins, and recent advances in peptide chemistry 14,31 have allowed this concept to be extended to larger proteins. Furthermore, our work highlights the need for strict compound characterization standards if results are to be reproducible and transferable. With the advancements in modern analytical techniques that are customary in synthetic organic chemistry (high-resolution mass spectrometry, NMR and X-ray structure determination) it has become necessary to apply the same rigorous standards to proteins and other biologics produced by recombinant expression.
Our study revealed, for the first time, human sSLURP-1 interactions with several neuronal nAChR subtypes (hα3β4, hα3β2, hα4β4 and hα9α10). We expect these findings will be important for understanding the in vivo function of SLURP-1 in human health and disease in the future.  ) were subsequently added. The pH was re-adjusted again to 6.9-7.0, and the mixture was stirred at room temperature for 10 h under an argon atmosphere. A fresh portion of TCEP (20 mM final concentration) was added and the mixture was stirred for another 20 min. The product was then filtered and purified by HPLC on a Phenomenex C18 column (22 × 250, 5 μm, 300 Å). It yielded 142 mg (16 μmol, 69%) of the fully reduced 81-mer polypeptide (>95% purity).

Methods
In vitro protein folding and disulfide formation. For in vitro folding and disulfide bond formation, 25 mg (2.8 μmol) of the purified and fully reduced peptide were dissolved in 5 mL of 6 M GdmHCl to give a concentration of 5 mg/mL. Folding was carried out at 4 °C and initiated by rapid 1:40 dilution of the peptide solution with folding buffer (100 mM Tris, 2.0 M urea, 0.5 M arginine, 4 mM reduced glutathione, 1 mM oxidized glutathione, adjusted to pH 8.0 at 4 °C with conc. HCl). The reaction was left at 4 °C and stirred for 3 days after which the mixture was acidified with TFA to give a pH of ~4, filtered and purified by HPLC on a Zorbax C3 column (10 × 250, 3 μm, 300 Å). The mass of synthetic SLURP-1 was determined by high-resolution ESI-MS on an AB SCIEX 5600 Triple-TOF mass spectrometer equipped with a nanoelectrospray ionization source. SLURP-1 observed mass 8837.1 ± 0.1 Da; calculated mass 8837.02 Da (monoisotopic mass). Isolated yield: 11.4 mg (1.3 μmol, 46%).
Scientific REPoRTS | 7: 16606 | DOI:10.1038/s41598-017-16809-0 NMR analysis. Synthetic SLURP-1(4 mg) was dissolved in 500 μL of 90% H 2 O/10% D 2 O solution and adjusted to pH 4.8 by adding 1 M NaOH. 2D 1 H-1 H TOCSY, NOESY as well as 1 H-15 N HSQC spectra were collected at 310 K using a 600 MHz Bruker spectrometer equipped with a cryogenically cooled probe. All spectra were recorded with an interscan delay of 1.0 s. The NOESY and TOCSY mixing times were 200 ms and 80 ms, respectively. Standard Bruker pulse sequences were used with WATERGATE for solvent suppression. NMR data were processed using Topspin (Bruker) and analyzed by CCPNMR 34 .
Molecular modeling. SLURP-1 was modeled by removing the N-terminal methionine residue from the PDB 2MUO structure using UCSF Chimera 35 . Both sSLURP-1 and rSLURP-1 structures were subjected to consequent equilibration 100 ns NVT (constant number of particles, volume and temperature) and 100 ns NPT (constant number of particles, pressure and temperature) ensemble simulations with constrained heavy atoms followed by 50 ns unconstrained molecular dynamics simulations using the GROMACS-5.0.4 package (reference temperature 310 K, 100 mM NaCl). Then, three individual frames from each of these two simulations were used as starting structures for 5 ns unconstrained molecular dynamics simulations to confirm reproducibility of results.
Oocyte two-electrode voltage clamp recording and data analysis. Electrophysiological recordings were carried out 2-7 days post cRNA microinjection. Two-electrode voltage clamp recording of X. laevis oocytes expressing human nAChRs was performed at room temperature (21-24 °C) using a GeneClamp 500B amplifier and pClamp9 software interface (Molecular Devices, Sunnyvale, CA, USA) at a holding potential −80 mV. For rat nAChRs, electrophysiological recordings were made using turbo TEC-03X amplifier (NPI Electronic, Germany) and WinWCP recording software (University of Strathclyde, UK), at a holding potential −60 mV. Voltage-recording and current-injecting electrodes were pulled from GC150T-7.5 borosilicate glass (Harvard Apparatus, Holliston, MA, USA) and filled with 3 M KCl, giving resistances of 0.3-1 MΩ.
Initially, oocytes were briefly washed with bath solution (ND96/ND115/Ba 2+ Ringer's solution) followed by 3 applications of ACh using a HPLC injector with a 50 µL sample loop. Washout with bath solution was done for 3 min between ACh applications. Oocytes were incubated with sSLURP-1 for 5 min with the perfusion system turned off, followed by co-application of ACh and sSLURP-1 with flowing bath solution. All sSLURP-1 solutions were prepared in ND96/ND115 + 0.1% bovine serum albumin (BSA), except for sSLURP-1 in Ba 2+ Ringer's solution. Peak current amplitudes before (ACh alone) and after (ACh + sSLURP-1) sSLURP-1 incubation were measured using Clampfit 10.7 software (Molecular Devices, Sunnyvale, CA, USA) or WinWCP software (University of Strathclyde, UK), where the ratio of ACh + sSLURP-1-evoked current amplitude to ACh alone-evoked current amplitude was used to assess the activity of sSLURP-1 at nAChRs. All electrophysiological data were pooled (n = 3 to 14) and represent means ± standard error of the mean (SEM). Data analysis was performed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA). Data sets were compared using unpaired two-tailed Student's t-test. Differences were regarded statistically significant when p < 0.05. The IC 50 was determined from concentration-response curve fitted to a non-linear regression function and reported with error of the fit.
Transfected Neuro2a cells were grown at 37 °C in 5% CO 2 -incubator for 48-72 h, then medium was removed and the cells were washed with external buffer containing (in mM) 140 NaCl, 2 CaCl 2 , 2.8 KCl, 4 MgCl 2 , 20 HEPES, 10 glucose at pH 7.4. Cells were pre-incubated with 5 μM sSLURP-1 for 20 min at room temperature before agonist addition (ACh or epibatidine (Tocris, UK)). To potentiate α7 nAChR response, PNU120596 (10 μM) was added to the pre-incubation solution. Cells were excited at 485 nm and emitted fluorescence was detected at 535 ± 10 nm, using a multimodal microplate reader Hidex Sense (Hidex, Turku, Finland). Fluorescence was recorded every 2 s for 3 min following agonist addition. Responses were measured as peak intensity minus basal fluorescence level, and are expressed as a percentage of a maximal response obtained to agonist. Data files were analyzed using HidexSence software (Hidex, Turku, Finland) and OriginPro 7.5 software (OriginLab, MA, USA, for statistical analysis). Negative controls were run in the presence of 4 μM α-Cbt.
Radioiodinated α-Bgt was added to a final concentration of 0.2 nM, and the mixture was incubated for 5 min. Binding was stopped by rapid filtration on double DE-81 filters (Whatman, Maidstone, UK) pre-soaked in binding buffer (for AChBPs) or GF/C filters (Whatman, Maidstone, UK) pre-soaked in 0.25% polyethylenimine (for GH 4 C 1 cells and T. californica electric organ membranes), unbound radioactivity was removed from the filters by washout (3 × 3 mL) with the binding buffer. Non-specific binding was determined in all cases using 3 h pre-incubation with 10 μM α-Cbt.
Data availability. All data generated during this study are included in the published article and the supplementary information file.